This project aims to investigate the process of vacuole formation in human erythrocytes and in their plasma membranes (erythrocyte ghosts). The two main goals are to investigate the mechanism of the process of vacuole formation and to compare erythrocytes from patients with hereditary spherocytosis (HS) with normal erythrocytes which have been treated to cause vacuole formation. HS, like sickle cell disease, is an hereditary disease of the erythrocyte; however, sickle cell disease is due to a defect in the hemoglobin, while HS appears to be due to a defect in the erythrocyte membrane. Many of the properties of HS erythrocytes resemble those of vacuolated erythrocytes, so that a closer investigation of the relationship between those two conditions is planned. Vacuole formation depends upon a supply of ATP both in whole erythrocytes and in ghosts. In ghosts, and probably in whole erythrocytes as well, vacuole formation is dependent upon hydrolysis of ATP by the Mg ions-dependent ATpase. The fundamental biochemistry of the human erythrocyte membrane is understood better than that of any other biological membrane, and is under continued intensive study. Thus, the situation is promising for an investigation of the detailed mechanism of vacuole formation in this membrane. In order to determine the way in which vacuole formation works, the effects of a number of treatments of the erythrocyte membrane will be considered. The effect of method of ghost preparation on vacuole formation will be systematically investigated. The effects of removal of various protein components and of sialic acid will be studied, and the role of spectrin will be investigated by various techniques. The relationship between the mobility of membrane proteins and the induction of vacuole formation will be investigated. By screening of inactivating agents, it may be possible to find one which is sufficiently specific that it will allow the identification of the proteins involved in the system responsible for vacuole formation.